Between gestational day E12 and 18, about half of the mitotic neuronal cells die in embryonal mouse forebrain due to apoptosis. Studies with caspase knockout mice have demonstrated that apoptosis is necessary to prevent hyperproliferation of neuronal stem cells and subsequent, severe brain malformation. It is not known, however, which factors selectively induce or prevent apoptosis in individual, differentiating neuronal stem cells. We have shown for the first time, that the peak time of apoptosis in embryonal mouse brain (E14.5) is concurrent with elevation of endogenous ceramide and activation of caspase 3. We have also shown that the concentration of ceramide is high enough to kill neuronal progenitor cells grown in culture. From these observations, we propose that elevation of ceramide may be critical for induction of apoptosis in differentiating neuronal stem cells. Recently, several studies have reported that the ceramide-mediated formation of an inhibitory complex between PAR-4 and atypical PKC ,induces apoptosis. Our own studies with in vitro differentiated embryonic stem (ES) cells have shown that induction of apoptosis by ceramide is concurrent with up-regulation of PAR-4. We propose that PAR-4 is one of probably several pro-apoptotic proteins that induce apoptosis when their expression and that of ceramide is elevated. Our main hypothesis is that simultaneous upregulation of endogenous ceramide and ceramide-associated proteins (CAPs) results in formation of a pro-apoptotic protein complex (PAC) that triggers apoptosis in mitotic neuronal stem cells by suppression of anti-apoptotic, cell survival signaling. We will test this hypothesis in three Specific Aims using murine ES cells as model system. In Specific Aim 1, we will test the hypothesis that up-regulation of ceramide induces apoptosis specifically in mitotic neuronal stem cells. In Specific Aim 2, we will test the hypothesis that ceramide/CAP-induced PAC formation results in activation of caspases. In Specific Aim 3, we will test the hypothesis that elevation of ceramide and down-regulation of cell survival signaling is synchronized by the cell cycle and growth factors. In conclusion, this study will identify mechanisms for regulation of apoptosis by ceramide that are critical for normal brain development and/or the etiology of subsequent, pathological disorders in adulthood.